Molecular identification and analysis of Borrelia burgdorferi sensu lato in lizards in the southeastern United States

Lyme borreliosis (LB) group spirochetes, collectively known as Borrelia burgdorferi sensu lato, are distributed worldwide. Wild rodents are acknowledged as the most important reservoir hosts. Ixodes scapularis is the primary vector of B. burgdorferi sensu lato in the eastern United States, and in the southeastern United States, the larvae and nymphs mostly parasitize certain species of lizards. The primary aim of the present study was to determine whether wild lizards in the southeastern United States are naturally infected with Lyme borreliae. Blood samples obtained from lizards in Florida and South Carolina were tested for the presence of LB spirochetes primarily by using B. burgdorferi sensu lato-specific PCR assays that amplify portions of the flagellin (flaB), outer surface protein A (ospA), and 66-kDa protein (p66) genes. Attempts to isolate spirochetes from a small number of PCR-positive lizards failed. However, PCR amplification and sequence analysis of partial flaB, ospA, and p66 gene fragments confirmed numerous strains of B. burgdorferi sensu lato, including Borrelia andersonii, Borrelia bissettii, and B. burgdorferi sensu stricto, in blood from lizards from both states. B. burgdorferi sensu lato DNA was identified in 86 of 160 (54%) lizards representing nine species and six genera. The high infection prevalence and broad distribution of infection among different lizard species at different sites and at different times of the year suggest that LB spirochetes are established in lizards in the southeastern United States.     

Regulation of Escherichia coli K5 capsular polysaccharide expression: Evidence for involvement of RfaH in the expression of group II capsules

Abstract We developed a quick typing method for Borrelia burgdorferi sensu lato strains using a fla gene-based PCR assay, followed by dot blot hybridization with non-radioactive species-specific probes. Thirty-six out of 46 strains belonged to one of the four described species ( B. burgdorferi sensu stricto n = 11, B. garinii n = 11, B. afzelii n = 9 and B. japonica n = 5) and hybridized with its own species-specific probe. Among the 10 remaining American strains, two new additional genomic groups were identified. This finding was confirmed by direct sequenching of the fla gene-derived amplicons and whole DNA hybridization.     

Substantial rise in the prevalence of Lyme borreliosis spirochetes in a region of western Germany over a 10-year period

More than a decade after a study on the transmission cycle of Borrelia burgdorferi sensu lato in the Siebengebirge, a nature reserve near Bonn, Germany, questing nymphal and adult Ixodes ricinus ticks were collected again in three selected areas of the same low mountain range and examined for infection with B. burgdorferi sensu lato. Between May and October 2001, a total of 1,754 ticks were collected by blanket dragging; 374 ticks were analyzed for B. burgdorferi sensu lato by both an immunofluorescence assay (IFA) and at least two different PCR tests, whereas 171 ticks were analyzed by PCR only. By combining all assays, an average of 14% of the ticks tested positive for B. burgdorferi sensu lato, 5.5, 15.8, and 21.8% in the three collection areas. Of the nymphs and adults examined, 12.9 and 21.1%, respectively, were found to be spirochete infected. A lower total infection prevalence was obtained by IFA (14.4%) than by a nested PCR approach (16.5%), but both were higher than that obtained by a simple PCR approach (11.9%). Compared with data collected over a decade ago, the mean infection prevalence of B. burgdorferi sensu lato in the ticks was significantly higher for all three biotopes, whereas a similar pattern of habitat-specific infection prevalence was observed. Genotyping of B. burgdorferi sensu lato revealed high relative prevalences of B. valaisiana (identified in 43.1% of infected ticks) and B. garinii (32.3%), whereas B. afzelii (12.3%) and B. burgdorferi sensu stricto (1.5%) were relatively rare. We conclude that B. burgdorferi sensu lato infection has increased in this region over the last 15 years due to presently unknown changes in ecological conditions, perhaps related to climate change or wildlife management.     

The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene

The 26 to 28 kb circular plasmid of B. burgdorferi sensu lato (cp26) is ubiquitous among bacteria of this group and contains loci implicated in the mouse–tick transmission cycle. Restriction mapping and Southern hybridization indicated that the structure of cp26 is conserved among isolates from different origins and culture passage histories. The cp26 ospC gene encodes an outer surface protein whose synthesis within infected ticks increases when the ticks feed, and whose synthesis in culture increases after a temperature upshift. Previous studies of ospC coding sequences showed them to have stretches of sequence apparently derived from the ospC genes of distantly related isolates by homologous recombination after DNA transfer. We found conservation of the promoter regions of the ospC and guaA genes, which are divergently transcribed. We also demonstrated that the increase in OspC protein after a temperature upshift parallels increases in mRNA levels, as expected if regulatory regions adjoin the conserved sequences in the promoter regions. Finally, we used directed insertion to inactivate the ospC gene of a non-infectious isolate. This first example of directed gene inactivation in B. burgdorferi shows that the OspC protein is not required for stable maintenance of cp26 or growth in culture.     

Local variations in the distribution and prevalence of Borrelia burgdorferi sensu lato genomospecies in Ixodes ricinus ticks.

Unfed nymphal and adult Ixodes ricinus ticks were collected from five locations within the 10,000-ha Killarney National Park, Ireland. The distribution and prevalence of the genomospecies of Borrelia burgdorferi sensu lato in the ticks were investigated by PCR amplification of the intergenic spacer region between the 5S and 23S rRNA genes and by reverse line blotting with genomospecies-specific oligonucleotide probes. The prevalence of ticks infected with B. burgdorferi sensu lato was significantly variable between the five locations, ranging from 11.5 to 28.9%. Four genomospecies were identified as B. burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, and VS116. Additionally, untypeable B. burgdorferi sensu lato genomospecies were identified in two nymphs. VS116 was the most prevalent of the genomospecies and was identified in 50% of the infected ticks. Prevalences of B. garinii and B. burgdorferi sensu stricto were similar (17 and 18%, respectively); however, significant differences were observed in the prevalence of these genomospecies in mixed infections (58.8 and 23.5%, respectively). Notably, the prevalence of B. afzelii was low, comprising 9.6 and 7.4%, respectively, of single and mixed infections. Significant variability was observed in the distribution and prevalence of B. burgdorferi sensu lato genomospecies between locations in the park, and the diversity and prevalence of B. burgdorferi sensu lato genomospecies was typically associated with woodland. The distributions of B. burgdorferi sensu lato genomospecies were similar in wooded areas and in areas bordering woodland, although the prevalence of B. burgdorferi sensu lato infection was typically reduced. Spatial distributions vegetation composition, and host cenosis of the habitats were identified as factors which may affect the distribution and prevalence of B. burgdorferi sensu lato genomospecies within the park.     

Distinct combinations of Borrelia burgdorferi sensu lato genospecies found in individual questing ticks from Europe

The genetic diversity of Borrelia burgdorferi sensu lato was assessed in individual adult Ixodes ricinus ticks from Europe by direct PCR amplification of spirochetal DNA followed by genospecies-specific hybridization. Analysis of mixed infections in the ticks showed that B. garinii and B. valaisiana segregate from B. afzelii. This and previous findings suggest that host complement interacts with spirochetes in the tick, thereby playing an important role in the ecology of Lyme borreliosis.     

Detection of Lyme disease spirochetes in the skin of naturally infected wild sika deer (Cervus nippon yesoensis) by PCR.

We demonstrated the presence of Borrelia burgdorferi sensu lato DNA in the skin tissues of naturally infected wild sika deer, using PCR. The risk of transmission of B. burgdorferi sensu lato is recognized in sika deer.     

Identification of a new Borrelia species among small mammals in areas of northern Spain where Lyme disease is endemic

The role of small mammals as reservoir hosts for Borrelia burgdorferi was investigated in several areas where Lyme disease is endemic in northern Spain. A low rate of infestation by Ixodes ricinus nymphs was found in the small mammal populations studied that correlated with the near-absence of B. burgdorferi sensu lato in 184 animals tested and with the lack of transmission of B. burgdorferi sensu lato to I. ricinus larvae that fed on them. In contrast, questing ticks collected at the same time and in the same areas were found to carry a highly variable B. burgdorferi sensu lato repertoire (B. burgdorferi sensu stricto, Borrelia garinii, Borrelia valaisiana, and Borrelia afzelii). Interestingly, the only isolate obtained from small mammals (R57, isolated from a bank vole) grouped by phylogenetic analyses with other Borrelia species but in a separate clade from the Lyme disease and relapsing fever organisms, suggesting that it is a new species. This new agent was widely distributed among small mammals, with infection rates of 8.5 to 12% by PCR. Moreover, a high seroprevalence to B. burgdorferi sensu lato was found in the animal sera, suggesting cross-reactivity between B. burgdorferi sensu lato and R57. Although small mammals do not seem to play an important role as reservoirs for B. burgdorferi sensu lato in the study area, they seem to be implicated in the maintenance of spirochetes similar to R57.     

Improved method of detection and molecular typing ofBorrelia burgdorferi sensu lato in clinical samples by polymerase chain reaction without DNA purification

A simple assay by polymerase chain reaction was used for the of detection of Borrelia burgdorferi, causative agent of Lyme borreliosis (LB). It involves no DNA purification and is based on the amplification of a specific region of ospA gene of B. burgdorferi, followed by direct detection of the PCR product with SYBR Green I by agarose gel electrophoresis. The method was used to analyze samples from patients with LB diagnosis, with presumable infection with the LB spirochete, those with unclear clinical symptoms and after the course of an antibiotic treatment. Spirochetal DNA was detected by PCR even in contaminated samples in which B. burgdorferi was overgrown by fungi and other bacteria. Spirochetal DNA was detected and borrelia species was identified in cerebrospinal fluid of two patients hospitalized with the diagnosis "fever of unknown origin". Western blot and ELISA were negative in both cases. Total analysis of 94 samples from the hospital in Ceské Budejovice (South Bohemia, Czechia) showed infection with B. burgdorferi sensu stricto in 11% and B. garinii in 15% of cases. The highest prevalence was found for B. afzelii (43%). Co-infection was confirmed in 24 % of the analyzed symplex; 7% of samples that were B. burgdorferi sensu lato positive gave no results in DNA amplification with B. burgdorferi sensu stricto-, B. garinii- and B. afzelii-specific primers. The proposed reliable, rapid, unexpensive and specific technique could form the basis of laboratory tests for routine detection and identification of Lyme-disease spirochete in different samples.     

Introduced Siberian chipmunks (Tamias sibiricus barberi) harbor more-diverse Borrelia burgdorferi sensu lato genospecies than native bank voles (Myodes glareolus)

Little attention has been given in scientific literature to how introduced species may act as a new host for native infectious agents and modify the epidemiology of a disease. In this study, we investigated whether an introduced species, the Siberian chipmunk (Tamias sibiricus barberi), was a potentially new reservoir host for Borrelia burgdorferi sensu lato, the causative agent of Lyme disease. First, we ascertained whether chipmunks were infected by all of the B. burgdorferi sensu lato genospecies associated with rodents and available in their source of infection, questing nymphs. Second, we determined whether the prevalence and diversity of B. burgdorferi sensu lato in chipmunks were similar to those of a native reservoir rodent, the bank vole (Myodes glareolus). Our research took place between 2006 and 2008 in a suburban French forest, where we trapped 335 chipmunks and 671 voles and collected 743 nymphs of ticks that were questing for hosts by dragging on the vegetation. We assayed for B. burgdorferi sensu lato with ear biopsy specimens taken from the rodents and in nymphs using PCR and restriction fragment length polymorphism (RFLP). Chipmunks were infected by the three Borrelia genospecies that were present in questing nymphs and that infect rodents (B. burgdorferi sensu stricto, B. afzelii, and B. garinii). In contrast, voles hosted only B. afzelii. Furthermore, chipmunks were more infected (35%) than voles (16%). These results may be explained by the higher exposure of chipmunks, because they harbor more ticks, or by their higher tolerance of other B. burgdorferi sensu lato genospecies than of B. afzelii. If chipmunks are competent reservoir hosts for B. burgdorferi sensu lato, they may spill back B. burgdorferi sensu lato to native communities and eventually may increase the risk of Lyme disease transmission to humans.     

A molecular marker for the identification of the zoonotic reservoirs of Lyme borreliosis by analysis of the blood meal in its European vector Ixodes ricinus.

The efficacy of the mitochondrially encoded cytochrome b gene as a molecular marker for the discrimination of the reservoir host species of the Lyme borreliosis spirochete, Borrelia burgdorferi sensu lato (s.l.), in its European vector Ixodes ricinus (Acari: Ixodidae) was determined. Degenerate PCR primers were designed which amplified orthologous regions of the cytochrome b gene in several animal species which act as B. burgdorferi s.l. reservoirs and hosts for I. ricinus. PCR products were amplified and characterized by hybridization and restriction fragment length polymorphism analysis. Restriction fragment length polymorphism analysis of a 638-bp PCR product with HaeIII and DdeI revealed unique restriction fragment profiles, which allowed the taxonomic identification of animals to the genus level. A system was devised for the detection of the larval host blood meal from the remnants in unfed nymphal I. ricinus ticks by nested PCR amplification. An inverse correlation was demonstrated between amplicon size and successful PCR amplification of host DNA from the nymphal stage of the tick. The stability of the cytochrome b product as a marker for the identification of the larval host species in the nymphal instar was demonstrated up to 200 days after larval ingestion (approximately 165 days after molting) by reverse line blotting with a host-specific probe. This assay has the potential for the determination of the reservoir hosts of B. burgdorferi s.l. by using extracts from the same individual ticks for both the identification of the host species and the detection of the Lyme borreliosis spirochete.     

Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Francisella tularensis and their co-infections in host-seeking Ixodes ricinus ticks collected in Serbia

To evaluate the prevalence rate of tick-borne bacterial pathogens, unfed adult Ixodes ricinus ticks were collected from vegetation in 2001, 2003, and 2004 at 18 localities throughout Serbia. A total of 287 ticks were examined by PCR technique for the presence of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and Francisella tularensis. The highest prevalence rate was that for B. burgdorferi sensu lato (42.5%), followed by A. phagocytophilum (13.9%) and F. tularensis (3.8%). The presence of five B. burgdorferi sensu lato genospecies, namely, B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. lusitaniae, and B. valaisiana was identified by restriction fragment length polymorphism (RFLP) analysis. The most frequent B. burgdorferi sensu lato genospecies was B. lusitaniae, followed by B. burgdorferi sensu stricto. Co-infection by B. burgdorferi sensu stricto and B. lusitaniae was frequently observed. Co-infection by B. burgdorferi sensu lato and A. phagocytophilum and co-infection by B. burgdorferi sensu lato and F. tularensis appeared in 24 ticks. Sequencing of p44/msp2 paralogs of Serbian A. phagocytophilum showed that they were unique and distinct from those of A. phagocytophilum in US and UK. This is the first report of B. garinii, B. lusitaniae, B. valaisiana, as well as A. phagocytophilum and F. tularensis infected ticks in Serbia. These findings indicate a public health threat in Serbia of tick-borne diseases caused by B. burgdorferi sensu lato, A. phagocytophilum and F. tularensis.     

Association of Borrelia garinii and B. valaisiana with songbirds in Slovakia

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.     

Murine splenocyte proliferation by porin of Shigella dysenteriae type 1 and inhibition of bacterial invasion of HeLa cell by anti-porin antibody

Abstract A highly sensitive nested polymerase chain reaction method was designed for the detection of a wide spectrum of strains from Borrelia burgdorferi sensu lato. This technique allows the detection of as little as 3 fg of total genomic DNA extracted and purified from pure cultures of the organism, this amount corresponds to less than 10 organisms. Two sets of primers homologous to conserved spots in the coding region of the hbb gene, encoding a conserved histone-like protein, were constructed. These were based on a multiple sequence alignment of 39 strains representing all the genomic groups described in B. burgdorferi sensu lato.     

Diversity of Borrelia burgdorferi sensu lato in Russian Far East

Thirty strains of Borrelia burgdorferi sensu lato have been isolated from Ixodes persulcatus ticks and from skin lesions of Lyme disease patients in the Russian Far East from 1997 to 2003. We amplified full-length outer surface protein A (ospA) gene of all strains. BLAST search and following phylogenetic analysis showed that strains form four well-defined groups. Four strains belong to Borrelia afzelii species. Other strains distributed into tree major groups, identified as Borrelia garinii. Indeed, based on the ospA gene comparison, phylogenetic relationship of these groups among each other does not differ from relationship among other previously defined groups inside B. burgdorferi sensu lato genogroup, such as B. afzelii or Borrelia bissettii. Further investigations of genetic and serologic properties of the strains belonging to those groups are required in order to clarify their taxonomic status.     

Biological aspects of Lyme disease spirochetes: unique bacteria of the Borrelia burgdorferi species group

Background: Borrelia burgdorferi sensu lato is a group of at least twelve closely related species some of which are responsible for Lyme disease, the most frequent zoonosis in Europe and the USA. Many of the biological features of Borrelia are unique in prokaryotes and very interesting not only from the medical viewpoint but also from the view of molecular biology. Methods: Relevant recent articles were searched using PubMed and Google search tools. Results and Conclusion: This is a review of the biological, genetic and physiological features of the spirochete species group, Borrelia burgdorferi sensu lato. In spite of a lot of recent articles focused on B. burgdorferi sensu lato, many features of Borrelia biology remain obscure. It is one of the main reasons for persisting problems with prevention, diagnosis and therapy of Lyme disease. The aim of the review is to summarize ongoing current knowledge into a lucid and comprehensible form.     

Improving the specificity of 16S rDNA-based polymerase chain reaction for detecting Borrelia burgdorferi sensu lato-causative agents of human Lyme disease

AIMS: 16S rDNA sequences of Borrelia burgdorferi sensu lato were aligned with the 16S rDNA sequences of Borrelia hermsii, Borrelia turicatae, and Borrelia lonestari in order to identify primers that might be used to more specifically identify agents of human Lyme disease in ticks in human skin samples. METHODS AND RESULTS: Standard polymerase chain reaction (PCR), using an oligonucleotide sequence, designated TEC1, was shown, in combination with a previously developed primer (LD2) to amplify strains of B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, but not the non-Lyme causing B. hermsii or B. turicatae. This primer pair, designated Bbsl, was successfully used to amplify B. burgdorferi sensu lato from skin biopsies of patients with Lyme disease symptoms as well as from Ixodes scapularis, Amblyomma americanum and Dermacentor variabilis ticks. CONCLUSIONS: The primer set Bbsl allows for the rapid detection and differentiation of B. burgdorferi sensu lato from non-Lyme disease-causing Borrelia species in ticks and human tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR primer set, Bbsl, will greatly facilitate detection of the causative agents of Lyme disease in infected ticks and human skin samples assisting in epidemiological studies, and potentially allowing for a more rapid diagnosis of the disease in patients.     

Whole genome sequence of an unusual Borrelia burgdorferi sensu lato isolate

Human Lyme disease is caused by a number of related Borrelia burgdorferi sensu lato species. We report here the complete genome sequence of Borrelia sp. isolate SV1 from Finland. This isolate is to date the closest known relative of B. burgdorferi sensu stricto, but it is sufficiently genetically distinct from that species that it and its close relatives warrant its candidacy for new-species status. We suggest that this isolate should be named "Borrelia finlandensis."     

The presence of Borrelia valaisiana-related genospecies in ticks and a rodent in Taiwan

A field survey was conducted to investigate the presence of Borrelia burgdorferi sensu lato (s.l.) in six counties of Taiwan. Spirochetes were successfully isolated from one rodent ear sample out of 485 rodent ears and 53 live, fed tick (Ixodes granulatus) samples. The spirochetes were confirmed to be B. burgdorferi s.l. by real-time PCR. In addition, 23 of 113 tick samples were tested positive for Borrelia DNA according to real-time PCR. The Borrelia isolate from the rodent and the 23 Borrelia DNA samples from the ticks were identified as B. valaisiana-related genospecies by phylogenetic analysis based on flagellin gene sequences. These findings suggest that the Borrelia valaisiana-related strains are maintained in a zoonotic cycle between tick vectors and reservoir hosts in Taiwan.     

Genetic characterization of pathogenic Borrelia, group A14S, isolated in Ukraine

Borrelia Ir-5215, isolated from ticks Ixodes ricinus in Ukraine (the Crimean autonomous region), was identified by the method of the polymorphism of the fragment length of the restriction amplicon of rRNA spacer region 5S-23S. Its Msel-restriction profile was relatively similar to that of B. afzelii. The sequencing of spacer region rrf (5S)-rrl (23S) and 16S rRNA gene, as well as the analysis of the similarity of nucleotide sequences, obtained in the course of these study, revealed the differences between Borrelia sp, lr-5215 and six European species of Borrelia burgdorferi sensu lato and a high level of similarity (more than 95.1% for 5S-23S rRNA and 99.4% for 16S rRNA gene) to three known representatives of genome group A14S (Borrelia spp. A14S, I-77 and PC-Rq17). This suggests that isolated Borrelia lr-5215 is a new representative of pathogenic B. burgdorferi sensu lato genome group A14S, which is spread, together with Central Europe, also in southern Ukraine.